Within this application several publications are referenced by arabic numerals within brackets. Full citations for these, and other, publications may be found at the end of the specification immediately preceding the claims. The disclosures of all these publications in their entireties are hereby expressly incorporated by reference into the present application for the purposes of indicating the background of the present invention and illustrating the state of the art.
Thyroid nodules represent a common problem brought to medical attention. Four to seven percent of the United States adult population (10-18 million) has palpable thyroid nodules, and up to 50% of American women older than age 50 have nodules visible by thyroid ultrasound [1]. The majority (>95%) of thyroid nodules are benign; however, malignancy risk increases with male gender, nodule size, rapid growth and associated symptoms, extremes of age (<30 and >60 years), underlying autoimmune disease (e.g. Graves' Disease), nodule growth under thyroid hormone suppression, personal or family history of thyroid malignancy, and radiation exposure [2].
Thorough history and physical examination, serum thyrotropin (TSH) level, thyroid ultrasound, and fine need aspiration biopsy (FNAB) are utilized to evaluate patients with thyroid nodules. Patients with thyroid nodules and normal or elevated serum TSH typically undergo thyroid ultrasound to determine if FNAB is warranted. Nodules with a maximum diameter greater than 1.0-1.5 cm with solid elements, or nodules of any size demonstrating suspicious features on ultrasound should undergo FNAB [3]. Given the increased risk of malignancy in thyroid incidentalomas detected by 18FDG-PET (Fluorodeoxyglucose or Fludeoxyglucose positron emission tomography) (14-50%) or sestamibi scan (22-66%), FNAB is indicated under these circumstances [4, 5]. Functioning thyroid nodules (suppressed TSH, hyperfunctioning on radionuclide scan) do not require FNAB in the absence of clinically suspicious findings.
Fine needle aspiration biopsy is a cost effective and accurate diagnostic tool for thyroid nodules. In experienced hands, the sensitivity and specificity of FNAB are very high, 95% and 99%, respectively, in positive and negative cases [6]. A six tiered classification system for FNAB is favored that is associated with increased risk of malignancy across the spectrum of: unsatisfactory or non-diagnostic specimen (unknown), benign (<1%), follicular lesion (atypia) of undetermined significance (5-10%), follicular neoplasm (20-30%), suspicious for malignancy (50-75%), and malignant (100%) [3]. Over 20% of patients undergoing FNAB of a thyroid nodule have indeterminate cytology (follicular neoplasm), and they require and are exposed to the function-limiting complications (impaired voice and swallowing) of thyroid lobectomy/isthmusectomy conducted purely for the purpose of attaining a more definitive diagnosis. Given that the majority of patients with follicular neoplasms have benign surgical pathology, thyroidectomy in these patients is conducted principally with diagnostic intent [7]. Electrical impedance scanning (EIS) is another tool for scanning thyroid nodules [9, 10]. Utilization of EIS can result in a significant reduction (67%) in the number of purely diagnostic thyroid resections for follicular neoplasms [8, 9].
Fine needle aspiration cytology has a high diagnostic accuracy and is a practicable test for the initial evaluation of thyroid nodules. However, the efficacy of FNA for the differential diagnosis of follicular and Hurthle cell neoplasms remains imperfect. As the majority of detected thyroid nodules are benign and cytology, even in the best of hands, is indeterminate in 20% of fine needle aspirates, the frequency of diagnostic or non-therapeutic thyroid resection is increasing.
As the majority of patients with indeterminate FNA cytology have benign nodules, surgical operations are undertaken primarily with diagnostic intent. Thus, it is difficult to non-invasively differentiate benign and clinically inconsequential low-risk malignant nodules from those that indeed stand to benefit from resection. Color Doppler sonography with quantitative analysis of tumor vascularity, in conjunction with conventional ultrasonographic assessment of echogenicity, halo, microcalcifications, and tumor size, may provide a means for differentiating malignant from benign solid thyroid nodules in the pre-operative setting [11-14]. However, the predictive value of this combined technique is achieved by compromising diagnostic sensitivity [15]. The predictive value of ultrasonography may be enhanced significantly through the application of ultrasound thyroid elastography [16-17]. The application of 18F-FDG PET shows high sensitivity for the diagnosis of malignancy in thyroid nodules demonstrating indeterminate cytology on pre-operative FNA. However, the low specificity of the technique limits its utility [18-19].
Cellular changes alter the flow of electrical current through living tissue, and differences in cellular electrical signature between malignant and non-malignant tissue has been identified and studied extensively since the 1920's [20]. EIS devices measure tissue impedance characteristics and identify irregularities in conductance and capacitance that are associated with increased levels of cellular activity and malignant transformation [21]. EIS measurements are obtained by introducing a known, low-level, biocompatible, alternating current to the body via a hand-held electrical signal generator. The signal is directed through the measured tissue and collected via a non-invasive surface probe. EIS is safe, feasible, and diagnostically accurate in detecting differences in the bioelectrical signature of benign and malignant tissue through body surface measurements of suspicious skin lesions and lymph nodes, and breast abnormalities [22-30]. EIS is a safe, rapid, realtime, and non-invasive imaging modality with a predictive value sufficient to make it an adjunct to FNA, particularly in the setting of indeterminate cytology [8, 9].
Recognizing that individual variables, though independently associated with thyroid cancer, are insufficient in predicting the risk of malignancy in any given thyroid nodule, multivariate predictive algorithms have been developed to determine the cumulative risk of malignancy for this clinical problem [10, 31]. One predictive algorithm utilizes a multivariate stepwise regression model to predict malignancy in thyroid nodules in a highly selected patient population on the basis of patient age, calcifications in a sonographically solid nodule, and FNAB cytology [10]. Another predictive algorithm applies multivariate modeling in patients with indeterminate thyroid nodules to define male gender, nodule size exceeding 4 cm, and character of the gland by palpation (dominant nodule in multi-nodular goiter) to predict the risk of thyroid malignancy [31]. The development of this predictive algorithm was limited to a narrow population of patients with follicular neoplasia by FNAB, and did not include imaging-based variables according to standard of practice in the predictive model.
Many electronic clinical decision support systems have been developed that rely on human expertise to develop decision-support rules rather than calculating a specific estimate of outcome using historical source data. Such “expert systems” take two forms. The first form is a system where clinical experts, following a systematic review of the literature, devise a system of static decision making rules for clinical decision support. The second form is a system where clinicians in the treating facility, usually basing their judgments on personal experience and the literature, devise a set of rules for clinical decision making in their own institution. The rules developed under both systems can either be implemented in publication format, in the form of published guidelines, or as a set of static decision rules in a clinical informatics system.
Transplant glomerulopathy (TG) is another disease that is difficult to diagnose. Transplant glomerulopathy is a distinctive lesion identified histologically on allograft biopsy and is associated with rapid decline in glomerular filtration rate and poor outcome. It is defined by a characteristic doubling of the glomerular basement membrane as well as increasing evidence that supports an immunologic pathogenesis; however, the molecular pathways involved have not been elucidated. Currently, transplant glomerulopathy must be diagnosed by microscopy, whether light or electron, at a minimum and thus necessitates an advanced disease stage, for which there is no cure.
Long-term kidney allograft function continues to improve modestly, despite dramatic improvements in acute rejection rates and short term patient and graft survivals. Measurement of serum creatinine is typically the primary monitoring modality following kidney transplantation. Significant changes in serum creatinine, and/or the development of proteinuria, result in a series of maneuvers to define the many potential etiologies of acute and chronic allograft dysfunction. Allograft biopsy is the current standard of these maneuvers, although morphologic analysis may not easily distinguish these etiologies. Furthermore, the analysis may be limited in regards to prognostic importance and functional outcome.
Gene expression analysis using microarrays and real-time polymerase chain reaction (PCR) has been applied broadly in the field of renal transplantation. Gene expression changes found in renal biopsies, urine sediment, and peripheral white blood cells have been used to evaluate allografts with stable function, acute rejection, and chronic allograft dysfunction. In addition, gene expression within the renal allograft pre-reperfusion or reperfusion periods has been correlated with delayed graft function and medium term allograft survival.
Several well-established relationships support that such an approach to identifying TG has biologic relevance. The relationship between pathology and cell signaling (chemokine expression), cell trafficking (adhesion molecule expression) and tissue remodeling (MMP expression) is supported by current models of TG. TG is believed to be secondary to binding of donor specific antibodies to endothelium with resulting stimulation and recruiting of secondary mediators leading to an inflammatory response. This inflammatory response and subsequent tissue injury has been associated with chemokine, adhesion molecule and MMP expression. Additionally, adhesion molecule expression has been shown to be associated with both chronic disease and stable function in renal transplant recipients. Alteration of chemokine expression has been linked to costimulatatory molecules (CD28, 40L, 80, 86) and IL-10 has been demonstrated to be elevated in allografts with stable function. The development of TG and Cd4 expression has also been well characterized.
The majority of modern war wounds are caused by blasts and high-energy ballistics [32-34]. Complex traumatic wounds require aggressive surgical care, including serial debridements to remove devitalized tissue and decrease bacterial load. Positive-pressure irrigation, negative-pressure and vacuum-assisted closure (VAC) have improved wound management [35-36]. However, despite these technological advances, the basic surgical decision regarding appropriate timing of surgical traumatic wound closure or coverage remains very subjective.
Poorly defined pathophysiology of acute wound failure partially contributes to the difficulties of objectively assessing wound healing. Current criteria for wound closure or coverage consider many subjective factors, which include the patient's general condition, injury location, adequacy of perfusion, and the gross appearance of the wound. Factors used to assess the patient's general condition include nutritional and nonspecific systemic inflammatory parameters. Relevance of injury location and visual assessment of the wound, such as the appearance of granulation tissue, are subjectively determined by the surgeon. Thus, there is considerable intra-observer variability in wound assessment. Furthermore, the decision making process used to make wound closure determination are ill-defined. After evaluating these factors, surgeons often reach a wound status determination base on his/her experience and discretion. Therefore, even in the hands of seasoned surgeons, some wounds ultimately fail. Unfortunately, other wounds with the biologic ability to heal will undergo unnecessary surgical debridements, adding treatment costs and exposing patients to additional anesthetic and surgical morbidity risk. Objective criteria and decision algorithms to define the appropriate timing of wound closure are needed.
The molecular landscape of the wound ultimately determines the fate of the wound healing process. Acute wounds typically heal by an interdependent sequence of events mediated by inflammatory messengers. The wound healing process generally has three phases. They are the inflammatory phase, the proliferative phase, and the maturational phase (or remodeling phase). The inflammatory phase is characterized by hemostasis and inflammation and typically lasts one to three days. After injury to tissue occurs, damaged cell membranes immediately release thromboxane A2 and prostaglandin 2-alpha, potent vasoconstrictors. This initial response helps to limit hemorrhage. After a short period, capillary vasodilatation occur secondary to local histamine release, and the cells responsible for inflammation are able to migrate to the wound bed. The timeline for cell migration in a normal wound healing process is predictable.
Platelets, the first response cell, release multiple chemokines, including epidermal growth factor (EGF), fibronectin, fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin, and von Willebrand factor. These factors help stabilize the wound through clot formation. They act to control bleeding and limit the extent of injury. Platelet degranulation also activates the complement cascade, specifically C5a, which is a potent chemoattractant for neutrophils.
As the inflammatory phase continues, more immune response cells migrate to the wound. Neutrophil, the second response cell, is responsible for debris scavenging, complement-mediated opsonization of bacteria, and bacteria destruction via oxidative burst mechanisms (superoxide and hydrogen peroxide formation). The neutrophils kill bacteria and decontaminate the wound from foreign debris.
The next cells present in the wound are the leukocytes and the macrophages (monocytes). Macrophage is essential for wound healing. Numerous enzymes and cytokines are secreted by the macrophage, including collagenases, which debride the wound; interleukins and tumor necrosis factor (TNF), which stimulate fibroblasts (production of collagen) and promote angiogenesis; and transforming growth factor (TGF), which stimulates keratinocytes. This marks the transition into the process of tissue reconstruction, the proliferative phase.
Epithelialization, angiogenesis, granulation tissue formation, and collagen deposition are the principal steps in the proliferative phase of wound healing. Epithelialization occurs early in wound repair. If the basement membrane remains intact, the epithelial cells migrate upwards in the normal pattern, as in first-degree skin burn. The epithelial progenitor cells remain intact below the wound, and the normal layers of epidermis are restored in 2-3 days. If the basement membrane has been destroyed, similar to a second- or third-degree burn, then the wound is reepithelialized from the normal cells in the periphery and from the skin appendages, if intact (eg, hair follicles, sweat glands).
Angiogenesis, stimulated by TNF-alpha, is marked by endothelial cell migration and capillary formation. The new capillaries deliver nutrients to the wound and help maintain the granulation tissue bed. The migration of capillaries into the wound bed is critical for proper wound healing. The granulation phase and tissue deposition require nutrients supplied by the capillaries, and failure for this to occur results in a chronically unhealed wound. Mechanisms for modifying angiogenesis are under study and have significant potential to improve the healing process.
The final part of the proliferative phase is granulation tissue formation. Fibroblasts differentiate and produce ground substance and then collagen. The ground substance is deposited into the wound bed. Collagen is then deposited as the wound undergoes the final phase of repair. Many different cytokines are involved in the proliferative phase of wound repair. The steps and the exact mechanism of control have not been elucidated. Some of the cytokines include PDGF, insulin like growth factor (IGF), and EGF. All are necessary for collagen formation.
The final phase of wound healing is the maturational phase. The wound undergoes contraction, ultimately resulting in a smaller amount of apparent scar tissue. The entire wound healing process is a dynamic continuum with an overlap of each phase and continued remodeling. Wound reaches maximal strength at one year and result in a tensile strength that is 30% of normal skin. Collagen deposition continues for a prolonged period, but the net increase in collagen deposition plateaus after 21 days.
Proper wound healing involves a complex interaction of cells and cytokines working in concert. Particularly, cytokines and chemokines orchestrate the progression of healing and are fundamental to the cellular and biochemical events that occur during acute wound healing. These effectors can be measured in serum and wound effluent using modern molecular techniques.
Currently, the only available commercial product proven to be efficacious in wound healing is PDGF, which is available as recombinant human PDGF-BB. In multiple studies, recombinant human PDGF-BB has been demonstrated to reduce healing time and improve the incidence of complete wound healing in stage III and IV ulcers. Other cytokines being studied for wound healing include TGF-beta, EGF, and IGF-1.
Breast carcinoma is the most commonly diagnosed cancer and the second leading cause of cancer-related mortality among women in the United States [50]. In 2009, there were over 192,000 estimated new cases of cancer of the breast, and over 40,000 disease-specific deaths [50]. Breast cancer-related mortality rates have steadily decreased over the past two decades, largely due to improved disease detection and therapy [51].
As breast cancer in younger (under age 40) women is infrequently diagnosed in the early stages utilizing current screening guidelines, improved cancer screening and detection methods are important in current research, particularly in younger, at-risk women [52]. Breast cancer in younger women typically has unfavorable prognostic characteristics associated with increased disease-specific mortality [53-55]. Younger women are not typically referred for periodic imaging unless they are identified as being “high risk” [56]. “At risk” younger women with significant family history or genetic factors are encouraged to undergo frequent clinical and annual breast imaging surveillance, and to consider chemoprevention.
While increased surveillance for “at risk” women may be beneficial, the value of this approach is restricted by the rarity of breast cancer due to known genetic risk factors [57, 58]. Over 90% of breast cancers are detected in women who are not identified as “high risk” [52]. Furthermore, screening mammography is generally less accurate in younger women and those with increased breast tissue density commonly encountered in women under age 40 [59]. The reduced sensitivity of mammography for dense breasts impacts age groups in which a “life saved” often results in “higher” personal and societal costs in terms of altered life expectancy and personal productivity [60].
MRI is being used increasingly as a screening modality in high-risk women with a significant family history of breast cancer, or BRCA1 or BRCA2 gene mutations resulting in lifetime risk of cancer exceeding 20% [61]. Hence, breast MRI is applied to a relatively small proportion of all women. MRI is unaffected by breast tissue density; however, the high cost, requirement for intravenous contrast administration, and variable specificity limit its feasibility for widespread population-based screening [62, 63].
Tamoxifen is considered in both pre- and post-menopausal women, and Raloxifene is considered in post-menopausal women, with lobular carcinoma in situ (LCIS) or with a 5-year breast cancer risk estimate of ≥1.66% (according to the Gail Model or the NCI Breast Cancer Risk Assessment Tool), in order to reduce the risk of estrogen receptor-positive (ER+) breast cancer [64]. In the NSABP P-1 study, Tamoxifen (20 mg/day for 5 years) consistently reduced the incidence of breast cancer by 49% in at-risk women across all study age and risk groups (women age 35-59 with a ≥1.66% risk, those ≥60, or with prior LCIS), thereby demonstrating the efficacy of chemoprevention for this disease [65]. The MORE, CORE, RUTH and NSABP STAR Trials demonstrated consistent significant reductions in ER+ breast cancer incidence in at-risk post-menopausal women [64]. Subsequent analyses of the NSABP P-1 study data suggested improved quality-adjusted survival and cost effectiveness when Tamoxifen was initiated as early as age 35 in at-risk (Gail Model 5-year risk ≥1.66%) women [66, 67].
Lifetime relative risk assessment tools (e.g., Gail model) are available to identify women over age 35 years who are at-risk for breast cancer. However, the predictive value of mathematical models to estimate breast cancer risk varies according to age, menopausal status, race/ethnicity, and family history of breast cancer. Instruments such as the Gail model are imperfect for identifying increased cancer risk in younger women [68]. Current risk prediction models estimate population, not individual levels of breast cancer risk. Currently, the only criterion generally used to identify high-risk young women who could benefit from chemoprevention is family/genetic history. The value of this risk estimation paradigm is limited by the rarity of breast cancer due to known gene mutations.